L-Tryptophan is an essential amino acid necessary for protein synthesis in mammalian cells. In addition, tryptophan is the precursor for the neurotransmitter serotonin, the hormone melatonin, and contributes to the synthesis of the coenzymes NAD and NADP. Abnormalities in serotonin production or signaling are related to depression, anxiety and substance abuse (Gingrich et al. (2001) Psychopharmacology (Berl) 155: 1-10). Melatonin is necessary for regulation of somatic day-night rhythm (Saper et al. (2005) Nature 437:1257-1263). Mammalian cells cannot synthesize L-tryptophan and depend on transport machinery for its uptake and protein turnover for its production. Identified transporter proteins that are involved in uptake of tryptophan in human cells are b0AT1 (Broer et al. (2005) Biochem. Soc. Trans. 33:233-236), b0,+AT (Feliubadalo et al. (1999) Nat. Genet. 23:52-57), TAT1 (Kim et al. (2002) Genomics 79:95-103), y+-LAT1 and y+-LAT2 (Pfeiffer et al. (1999) EMBO J. 18:49-57; Torrents et al. (1998) J. Biol. Chem. 273:32437-32445), LAT1, LAT2, LAT3 and LAT4 (Babu et al. (2003) J. Biol. Chem. 278:43838-43845; Bodoy et al. (2005) J. Biol. Chem. 280:12002-12011; Rossier et al. (1999) J. Bio. Chem. 274:34948-34954; Verrey et al. (2003) Eur. J. Physiol. 44:529-533). Of these, b0,+AT, LAT1, LAT2, y+-LAT1, and y+-LAT2 are amino acid exchangers; they swap an internal amino acid molecule for an external one.
Tryptophan can be degraded through the kynurenine (or kynurenin) pathway for the biosynthesis of niacin. The rate-limiting step in this pathway is the opening of the indole ring by indoleamine 2,3-dioxygenase (IDO). Since the discovery that inhibition of IDO induced fetal allograft rejection in mice, the immunosuppressive function of tryptophan catabolism has been well established (Munn et al. (1998) Science 281:1191-1193). One proposed mechanism for the observed immunosuppression is the local depletion of tryptophan, which inhibits adaptive T-cell responses by forcing them into growth arrest and inducing apoptosis (Mellor et al. (2003) Adv. Exp. Med. Biol. 527). As such, the immune escape of many cancer cell types correlates with upregulated IDO expression and can in some cases be overcome by IDO inhibition (Muller et al. (2005) Nat. Med. 11:312-319). In addition, products of the kynurenine pathway are immunosuppressive themselves and may provide leads for the treatment of autoimmune disorders such as multiple sclerosis (Platten et al. (2005) Science 310:850-855). The transport machinery for the displacement of kynurenines across the cell membrane is not known.
Traditionally, cellular uptake of molecules has been determined using radiolabeled substrates, and levels have been measured in cell extracts via liquid chromatography or gas chromatography/mass spectrometry. Both methods are neither time-resolved nor specific, and lack high temporal or cellular/subcellular resolution. Tryptophan is also aromatic, and binds to many molecules non-specifically. Given the importance of L-tryptophan for human health, an analytical tool for non-invasive, time-resolved determination of intracellular L-tryptophan levels was deemed highly desirable.
Fluorescent indicator proteins (FLIPs) have been successful tools for real-time monitoring of metabolite levels in living cells. Typically, the nanosensors consist of a ligand-sensing domain, allosterically coupled to a pair of green fluorescent protein variants capable of resonance energy transfer, referred to as Förster Resonance Energy Transfer (FRET) or fluorescence resonance energy transfer. FRET efficiency depends on the distance between and relative orientation of the dipoles of the fluorophores. Ligand-binding induced conformational changes in the sensors result in altered FRET efficiencies, which correlate with the levels of the respective metabolites. Periplasmic binding proteins (PBPs) have been successfully exploited for the construction of FLIPs for imaging of key metabolites such as glucose (Fehr et al. (2003) J. Biol. Chem. 278:19127-19133) maltose (Fehr et al. (2002) Proc. Natl. Acad. Sci. USA 99:9846-9851), ribose (Lager et al. (2003) FEBS Lett. 553:85-89) and glutamate (Okumoto et al. (2005) Proc. Natl. Acad. Sci. USA 102:8740-8745). However, no tryptophan-binding PBPs have been described to date, thus an alternative ligand-sensing scaffold was explored for construction of a tryptophan nanosensor.
In γ-proteobacteria like Escherichia coli, transcription of the tryptophan biosynthetic operon is regulated by attenuation (Yanofsky (1981) Nature 289:751-758), and by the inhibitory binding of the tryptophan-activated repressor protein, TrpR, to the trp operator (Joachimiak et al. (1983) Proc. Natl. Acad. Sci. USA 80:668-672). Binding of L-tryptophan to the repressor results in conformational changes that enhance the repressor's affinity for the operator sequence (Zhang et al. (1987) Nature 327:591-597).